Biomedical subjects

Tor Ny

Publications and source records attributed to Tor Ny.

15 recordsLinked to original sources

PubMed2006

Functional corpora lutea are formed in matrix metalloproteinase inhibitor-treated plasminogen-deficient mice.

Corpus luteum (CL) formation involves dramatic tissue remodeling and angiogenesis. To determine the functional roles of the plasminogen activator and matrix metalloproteinase (MMP) systems in these processes, we have studied CL formation and function in plasminogen (plg)-deficient mice, with or without treatment with the broad-spectrum synthetic MMP inhibitor galardin. Both the adult pseudopregnant CL model and the gonadotropin-primed immature mouse model were used. We found that CL formed normally not only in plasminogen-deficient mice and in galardin-treated wild-type mice, but also in galardin-treated plg-deficient mice, suggesting that neither of the plasminogen activator and MMP systems is essential for CL formation. Nevertheless, in plg-deficient mice, serum progesterone levels were reduced by approximately 50%, and the progesterone levels were not reduced further by galardin treatment. When CL from plg-deficient mice were stained for several molecular markers for CL development and regression, they appeared healthy and vascularized, and were indistinguishable from CL from wild-type mice. This implies that the reduced progesterone levels were not caused by impaired CL formation. Taken together, our data suggest that neither plasmin nor MMPs, alone or in combination, are required for CL formation. Therefore, the tissue remodeling and angiogenesis processes during CL formation may be mediated by redundant protease systems. However, the reduced serum progesterone levels in plg-deficient mice suggest that plasmin, but not MMPs, plays a role in maintenance of luteal function. This role may be performed through proteolytic activation of growth factors and other paracrine factors.

Animals↗

PubMed2006

Spontaneous development of otitis media in plasminogen-deficient mice.

Inflammatory conditions of the ear, otitis media, are one of the most common disease entities in children. In this study, the role of the plasminogen (plg)/plasmin system for the spontaneous development of chronic otitis media was investigated by the analysis of plg-deficient mice. Whereas essentially all of the wild-type control mice kept a healthy status of the middle ear, all the plg-deficient mice gradually developed chronic otitis media with various degrees of inflammatory changes during an 18-week observation period. Five bacterial strains were identified in materials obtained from the middle ear cavities of six plg-deficient mice. Morphological studies revealed the formation of an amorphous mass tissue and inflammatory changes in the middle ears of plg-deficient mice. Immunohistochemical studies further indicate a mass infiltration of neutrophils and macrophages as well as the presence of T and B cells in the middle ear mucosa of these mice. Extensive fibrin deposition and an abnormal keratin formation were also observed in the tympanic membrane, the middle ear cavity and external ear canal in these mice. These results suggest that plg plays an essential role in protecting against the spontaneous development of chronic otitis media. Our findings also suggest the possibility of using plg for clinical therapy of certain types of otitis media.

Animals↗

PubMed2006

Successful ovulation in plasminogen-deficient mice treated with the broad-spectrum matrix metalloproteinase inhibitor galardin.

Many studies have suggested the hypothesis that the plasminogen activator (PA) system and the matrix metalloproteinase (MMP) system, either separately or in combination, may provide the proteolytic activity that is required for rupture of the follicular wall at the time of ovulation. Our recent studies on ovulation in plasminogen (plg)-deficient mice have, however, shown that plasmin is not required for normal ovulation, leading us to the hypothesis that MMPs may be a more important source of proteolysis for this process. To investigate the role of MMPs and also the possibility of a functional overlap or synergy between the MMP and PA systems during ovulation, we have studied ovulation efficiency in wild-type and plg-deficient mice treated with the broad-spectrum MMP inhibitor galardin. We found that in both wild-type mice and heterozygous plg-deficient (plg+/-) mice that had been treated with galardin prior to ovulation, there was a mild (18-20%) reduction in ovulation efficiency. Surprisingly, galardin treatment of plg-deficient (plg-/-) mice only caused an additional 14% reduction in ovulation efficiency as compared to vehicle-treated plg-/- mice. Our data therefore suggest that although MMPs may play a role in degradation of the follicular wall, they may not be obligatory for ovulation. In contrast to previous studies on tissue remodeling during wound healing and placental development, we have demonstrated that there is no obvious functional overlap or synergy between the PA and MMP systems, which has previously been thought to be essential for the ovulatory process.

Animals↗

PubMed2006

Plasmin/plasminogen is essential for the healing of tympanic membrane perforations.

Plasminogen has been proposed to play an important role in different tissue remodeling processes such as wound healing and tissue regeneration after injuries. The healing of tympanic membrane perforations is a well-organized chain of inflammatory events, with an initial invasion of inflammatory cells followed by reparative and restoration phases. Here we show that the healing of tympanic membrane perforations is completely arrested in plasminogen-deficient mice, with no signs of any healing even 143 days after perforation. Inflammatory cells were recruited to the wounded area, but there were no signs of tissue debridement. In addition, removal of fibrin, keratinocyte migration and in-growth of connective tissue were impaired. This contrasts with skin wound healing, where studies have shown that, although the healing process is delayed, it reaches completion in all plasminogen-deficient mice. Our finding that keratinocyte migration and re-epithelialization were completely arrested in plasminogen-deficient mice indicates that plasminogen/plasmin plays a more profound role in the healing of tympanic membrane perforations than in the healing of other epithelial wounds.

Animals↗

PubMed2005

Contrasting roles of plasminogen deficiency in different rheumatoid arthritis models.

OBJECTIVE: To investigate the contrasting roles of plasminogen deficiency between models of collagen-induced arthritis (CIA) and antigen-induced arthritis (AIA). METHODS: We developed a new animal model of arthritis, which we have called local injection-induced arthritis (LIA). In this model, we replaced methylated bovine serum albumin, which is normally used as an immunogen and is injected intraarticularly into the knee joint, with type II collagen (CII) to induce AIA. The severity of CIA, LIA, and AIA in wild-type and plasminogen-deficient mice was evaluated by clinical scoring or histologic grading. Necrosis was determined by histology and immunohistochemistry. RESULTS: After CII immunization alone, wild-type mice developed arthritis in most of the paws as well as in the knee joints, whereas plasminogen-deficient mice were totally resistant to the disease. Local knee injections of CII or saline slightly enhanced the severity of the knee arthritis in wild-type mice during a 60-day experimental period. Unexpectedly, the plasminogen-deficient mice also developed arthritis in joints that were injected with CII or saline. However, the arthritis was milder than that in their wild-type littermates. Sustained tissue necrosis was found only in the plasminogen-deficient mice after the local injection. CONCLUSION: Our data show that both the antigen and the joint trauma caused by the local injection are critical to explaining the contrasting roles of plasminogen deficiency in CIA and AIA. This further indicates that CIA and AIA have distinct pathogenic mechanisms. The data also suggest that plasmin may be required for the induction of these arthritis models that are critically dependent on complement activation.

Animals↗

PubMed2005

The plasminogen activator/plasmin system is essential for development of the joint inflammatory phase of collagen type II-induced arthritis.

The plasminogen activator (PA) system has been proposed to have important roles in rheumatoid arthritis. Here we have used the autoimmune collagen type II (CII)-induced arthritis (CIA) model and mice deficient for urokinase-type PA (uPA) or plasminogen to investigate the role of the PA system for development of arthritis. Our data revealed that uPA-deficient mice have a lower severity and incidence of CIA than wild-type mice. Furthermore, although >80% of wild-type control mice developed CIA, we found that none of the 50 plasminogen-deficient littermates that were tested developed CIA within a 40-day period. Antibody generation after CII immunization as well as the binding of labeled anti-CII antibodies to the surface of cartilage were similar in wild-type and plasminogen-deficient mice. No sign of inflammation was seen when plasminogen-deficient mice were injected with a mixture of monoclonal antibodies against CII. However, after daily injections of human plasminogen, these mice developed arthritis within 5 days. Our finding that infiltration of inflammatory cells into the synovial joints was impaired in plasminogen-deficient mice suggests that uPA and plasminogen are important mediators of joint inflammation. Active plasmin is therefore essential for the induction of pathological inflammatory joint destruction in CIA.

Animals↗

PubMed2004

Structural bases of the redox-dependent conformational switch in the serpin PAI-2.

Depending on the redox-status, the serpin plasminogen activator inhibitor type 2 (PAI-2) can exist in either a stable monomeric or polymerogenic form. The latter form, which spontaneously forms loop-sheet polymers, has an open beta-sheet A and is stabilized by a disulfide bond between C79 (in the CD-loop) and C161 (at the bottom of PAI-2). Reduction of this bond results in a closing of the beta-sheet A and converts PAI-2 to a stable monomeric form. Here we show that the stable monomeric and polymerogenic forms of PAI-2 are fully interconvertible, depending on redox-status of the environment. Our intramolecular distance measurements indicate that the CD-loop folds mainly on one side of the stable monomeric form of the inhibitor. However, the loop can translocate about 54A to the bottom of PAI-2 so that the C79-C161 disulfide bond can form under oxidizing conditions. We show also that the redox-active C79 can form a disulfide-link to the matrix protein vitronectin, suggesting that vitronectin can stabilize active PAI-2 in extracellular compartments. PAI-2 is therefore a rare example of a redox-sensitive protein for which the activity and polymerization ability are regulated by reversible disulfide bond formation leading to major translocation of a loop and significant conformational changes in the molecule.

Cysteine↗

PubMed2004

Residues determining the binding specificity of uncompetitive inhibitors to tissue-nonspecific alkaline phosphatase.

UNLABELLED: Recent data have pointed to TNALP as a therapeutic target for soft-tissue ossification abnormalities. Here, we used mutagenesis, kinetic analysis, and computer modeling to identify the residues important for the binding of known ALP inhibitors to the TNALP active site. These data will enable drug design efforts aimed at developing improved specific TNALP inhibitors for therapeutic use. INTRODUCTION: We have shown previously that the genetic ablation of tissue-nonspecific alkaline phosphatase (TNALP) function leads to amelioration of soft-tissue ossification in mouse models of osteoarthritis and ankylosis (i.e., Enpp1-/- and ank/ank mutant mice). We surmise that the pharmacologic inhibition of TNALP activity represents a viable therapeutic approach for these diseases. As a first step toward developing suitable TNALP therapeutics, we have now clarified the residues involved in binding well-known uncompetitive inhibitors to the TNALP active site. MATERIALS AND METHODS: We compared the modeled 3D structure of TNALP with the 3D structure of human placental alkaline phosphatase (PLALP) and identified the residues that differ between these isozymes within a 12 A radius of the active site, because these isozymes differ significantly in inhibitor specificity. We then used site-directed mutagenesis to substitute TNALP residues to their respective homolog in PLALP. In addition, we mutagenized most of these residues in TNALP to Ala and the corresponding residues in PLALP to their TNALP homolog. All mutants were characterized for their sensitivity toward the uncompetitive inhibitors l-homoarginine (L-hArg), levamisole, theophylline, and l-phenylalanine. RESULTS AND CONCLUSIONS: We found that the identity of residue 108 in TNALP largely determines the specificity of inhibition by L-hArg. The conserved Tyr-371 is also necessary for binding of L-hArg. In contrast, the binding of levamisole to TNALP is mostly dependent on His-434 and Tyr-371, but not on residues 108 or 109. The main determinant of sensitivity to theophylline is His-434. Thus, we have clarified the location of the binding sites for all three TNALP inhibitors, and we have also been able to exchange inhibitor specificities between TNALP and PLALP. These data will enable drug design efforts aimed at developing improved, selective, and drug-like TNALP inhibitors for therapeutic use.

Alkaline Phosphatase↗

PubMed2004

The reactive-center loop of active PAI-1 is folded close to the protein core and can be partially inserted.

Plasminogen activator inhibitor 1 (PAI-1) is the main inhibitor of plasminogen activators and plays an important role in many pathophysiological processes. Like other members of the serpin family, PAI-1 has a reactive center consisting of a mobile loop (RCL) with P1 and P1' residues acting as a "bait" for cognate protease. In contrast to the other serpins, PAI-1 loses activity by spontaneous conversion to an inactive latent form. This involves full insertion of the RCL into beta-sheet A. To search for molecular determinants that could be responsible for conversion of PAI-1 to the latent form, we studied the conformation of the RCL in active PAI-1 in solution. Intramolecular distance measurements by donor-donor energy migration and probe quenching methods reveal that the RCL is located much closer to the core of PAI-1 than has been suggested by the recently resolved X-ray structures of stable PAI-1 mutants. Disulfide bonds can be formed in double-cysteine mutants with substitutions at positions P11 or P13 of the RCL and neighboring residues in beta-sheet A. This suggests that the RCL may be preinserted up to residue P13 in active PAI-1, and possibly even to residue P11. We propose that the close proximity of the RCL to the protein core, and the ability of the loop to preinsert into beta-sheet A is a possible reason for PAI-1 being able to convert spontaneously to its latent form.

Animals↗

PubMed2003

Tissue-type plasminogen activator and its inhibitor plasminogen activator inhibitor type 1 are coordinately expressed during ovulation in the rhesus monkey.

Ovulation is a gonadotropin-controlled process that is essential for the propagation of all mammalian species. In the present study, we used a pregnant mare serum gonodotropin/human chorionic gonadotropin (hCG)-induced, synchronized ovulation model in rhesus monkeys and systematically investigated the roles of the plasminogen activator (PA) system in the ovulatory process of the primate. At different follicular developmental stages throughout the periovulatory period, samples of ovaries, granulosa cells, and theca-interstitial cells as well as follicular fluid were collected, and levels of PA and PA inhibitor type-1 (PAI-1) were evaluated by fibrin overlay, reverse fibrin overlay, Northern blot analysis, and in situ hybridization, respectively. We showed that in response to an injection of ovulation-triggering hCG, which mimics the preovulatory surge of LH in the circulation, granulosa cell-derived tissue-type PA (tPA) was substantially elevated in preovulatory follicles and reached its maximum level just before ovulation. Although theca-interstitial cell-derived PAI-1 was also stimulated by pregnant mare serum gonodotropin and hCG treatments, however, the maximum level of PAI-1 appeared 12 h earlier than that of tPA. When ovulation approached, accompanying the highest tPA level in the preovulatory follicles, the follicular PAI-1 level declined dramatically to its minimum value. Moreover, our data on the expression of follicular PA and PAI-1 over the periovulatory period were reinforced by results obtained at the mRNA level. Our data suggest that the coordinated expression of tPA and PAI-1 may be of importance for the follicular rupture process during ovulation in the primate.

Aging↗

PubMed2003

Expression pattern and functional studies of matrix degrading proteases and their inhibitors in the mouse corpus luteum.

The formation of the corpus luteum (CL) is accompanied with angiogenesis and tissue remodeling and its regression involves tissue degradation. Matrix degrading proteases such as plasminogen activators (PAs) and matrix metalloproteinases (MMPs) are thought to play important roles in such controlled proteolytic processes. In this study, in situ hybridization has been used to examine the regulation and expression pattern of mRNAs coding for proteases and protease inhibitors belonging to the PA- and MMP-systems during the life cycle of the CL in an adult pseudopregnant mouse model. Of the nine proteases and five protease inhibitors that were studied, the majority were found to be temporally expressed during the formation and/or the regression of the CL. However, the mRNAs coding for urokinase type PA (uPA), membrane-type 1 MMP (MT1-MMP), and tissue inhibitor of metalloproteinases type-3 (TIMP-3) were constantly expressed in the mouse CL throughout its whole life span. To study the functional role of uPA in the CL, we analyzed luteal formation and function in uPA deficient mice. Our results revealed no significant difference in ovarian weight, serum progesterone levels, and blood vessel density in the functional CL between uPA deficient and wild type control mice. The temporal and spatial expression pattern of proteases and protease inhibitors during the CL life span suggests that members of the PA- and MMP-systems may play important roles in the angiogenesis and tissue remodeling processes during CL formation, as well as in the tissue degradation during luteal regression. However, the absence of reproductive phenotypes in mice lacking uPA and several other matrix degrading proteases indicates that there are redundancies among different matrix degrading proteases or that tissue remodeling in the ovary may involve other additional unique elements.

Animals↗

PubMed2003

A redox-sensitive loop regulates plasminogen activator inhibitor type 2 (PAI-2) polymerization.

Plasminogen activator inhibitor type 2 (PAI-2) is the only wild-type serpin that polymerizes spontaneously under physiological conditions. We show that PAI-2 loses its ability to polymerize following reduction of thiol groups, suggesting that an intramolecular disulfide bond is essential for the polymerization. A novel disulfide bond was identified between C79 (in the CD-loop) and C161 (at the bottom of helix F). Substitution mutants in which this disulfide bond was broken did not polymerize. Reactive center loop peptide insertion experiments and binding of bis-ANS to hydrophobic cavities indicate that the C79-C161 disulfide bond stabilizes PAI-2 in a polymerogenic conformation with an open A-beta-sheet. Elimination of this disulfide bond causes A-beta-sheet closure and abrogates the polymerization. The finding that cytosolic PAI-2 is mostly monomeric, whereas PAI-2 in the secretory pathway is prone to polymerize, suggests that the redox status of the cell could regulate PAI-2 polymerization. Taken together, our data suggest that the CD-loop functions as a redox-sensitive switch that converts PAI-2 between an active stable monomeric and a polymerogenic conformation, which is prone to form inactive polymers.

Cysteine↗

PubMed2003

The spontaneous polymerization of plasminogen activator inhibitor type-2 and Z-antitrypsin are due to different molecular aberrations.

The wild-type form of plasminogen activator inhibitor type-2 (PAI-2) and the pathogenic Z-mutant of alpha(1)-antitrypsin (alpha(1)AT) are serpins that spontaneously polymerize by the loop-sheet mechanism. Compared to the consensus serpin sequence, both PAI-2 and Z-alpha(1)AT have deviations in the so-called breach region located at the top of the A beta-sheet. In the case of Z-alpha(1)AT, conformational perturbations caused by a single amino acid substitution result in polymerization in vivo and predisposes to disease. To test whether the polymerization of PAI-2 is due to aberrations in the breach region, we constructed substitution mutants of PAI-2 with conserved residues in this region. Analysis of the mutants revealed that deviations in the breach region modulate but are not the major cause of PAI-2 polymerization. Rather, PAI-2 exists in a highly polymerogenic conformation and does not require conformational rearrangements before polymerization can take place.

Hydrogen-Ion Concentration↗

PubMed2002

Matrix remodeling in the ovary: regulation and functional role of the plasminogen activator and matrix metalloproteinase systems.

In each reproductive cycle, extensive tissue remodeling takes place in the ovary during follicular development, ovulation, formation and regression of corpus luteum (CL) and follicular atresia. Several lines of indirect evidence suggest that these changes are mediated, in part, by proteases belonging to the plasminogen activator (PA) and the matrix metalloproteinase (MMP) systems. These two enzyme systems include both proteinases and associated inhibitors, that are thought to act in concert via a cascade of proteolytic events, the end result of which is the generation of a broad spectrum proteolytic activity, that can mediate physiological tissue remodeling throughout the body. The current review highlights the key features of these two enzyme systems and focuses on their regulation and functional role during the dynamic remodeling processes that takes place in the ovary during each reproductive cycle.

Animals↗

PubMed2002

Dimers of dipyrrometheneboron difluoride (BODIPY) with light spectroscopic applications in chemistry and biology.

A ground-state dimer (denoted D(I)) exhibiting a strong absorption maximum at 477 nm (epsilon = 97 000 M(-1)cm(-1)) can form between adjacent BODIPY groups attached to mutant forms of the protein, plasminogen activator inhibitor type 1 (PAI-1). No fluorescence from excited D(I) was detected. A locally high concentration of BODIPY groups was also achieved by doping lipid phases (micelles, vesicles) with BODIPY-labeled lipids. In addition to an absorption band located at about 480 nm, a new weak absorption band is also observed at ca. 570 nm. Both bands are ascribed to the formation of BODIPY dimers of different conformation (D(I) and D(II)). Contrary to D(I) in PAI-1, the D(II) aggregates absorbing at 570 nm are emitting light observed as a broad band centered at about 630 nm. The integrated absorption band of D(I) is about twice that of the monomer, which is compatible with exciton coupling within a dimer. The Förster radius of electronic energy transfer between a BODIPY excited monomer and the ground-state dimer (D(I)()) is 57 +/- 2 A. A simple model of exciton coupling suggests that in D(I) two BODIPY groups are stacked on top of each other in a sandwich-like configuration with parallel electronic transition dipoles. For D(II) the model suggests that the S(0) --> S(1) transition dipoles are colinear. An explanation for the previously reported (J. Am. Chem. Soc. 1994, 116, 7801) exceptional light spectroscopic properties of BODIPY is also presented. These are ascribed to the extraordinary electric properties of the BODIPY chromophore. First, changes of the permanent electric dipole moment (Delta(mu) approximately -0.05 D) and polarizability (-26 x 10(-40) C m(2) V(-1)) between the ground and the first excited states are small. Second, the S(0) <--> S(1) electronic transition dipole moments are perpendicular to Delta(mu).

Boron Compounds↗